The immune system consists of two major arms: innate and adaptive. Innate immune cells, in particular dendritic cells (DC), are critical in early responses to control pathogens, and play an essential role in activating the adaptive response. The later is mediated by lymphocytes, and is important in dealing with acute threats as was as secondary re-exposures (e.g. a second infection with the same organism). Upon primary exposure to antigen, nave T cells bearing complementary antigen receptors (TCRs) undergo rapid activation and clonal expansion, leading to the generation of effector T cells. The vast majority (90-95%) of antigen-specific effector T cells that participate in the primary immune response undergo apoptosis (cell death) after antigen clearance. A small subset, however, survives and gives rise to long-lived memory T cells. Upon antigen re-encounter, memory T cells respond swiftly and robustly to eliminate the pathogen. Based on their tissue distribution, cell surface markers, and effector functions, memory T cells have been divided into two major subsets. Memory T cells expressing receptors such as CD62L and CCR7, which allow efficient homing to lymph nodes (LN), are termed central memory (TCM) cells; memory T cells lacking LN-homing receptors and preferentially residing in non-lymphoid tissues are termed effector memory (TEM) cells. Notably, both memory subsets display high levels of the marker CD44. Once established CD4 and CD8 memory T cell populations can be maintained for many years in vivo. Although early studies suggested that continued antigenic stimulation is required for maintaining T cell memory, neither cognate antigen nor MHC-encoded molecules appear to be required for the long-term survival of CD4 or CD8 memory T cells. Members of the tumor necrosis factor receptor (TNFR) family are involved in T cell costimulation and play a role in T cell survival. Mice deficient in OX40, CD27, or 4-1BB show greater defects in secondary compared to primary responses when infected with pathogens such as lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus, and influenza. We developed a method of separating naive from memory T cells based upon their density. Using this technique, we made the following findings (especially with regard to CD8 T cell memory, which is important in anti-viral and anti-neoplastic responses): 1. Memory T cells exist in the G1 phase (high RNA, haploid DNA) of the cell cycle, whereas naive T cells exist in G0 (low RNA, haploid DNA). Stimulation of the former results in rapid release of high amounts of cytokines such as interferon gamma (IFNgamma). 2. Culture of purified memory T cells in the absence of other cells (resting) allows them to revert to G0. When they do, they respond to stimulation to the same degree and with the same kinetics as naive T cells. Culture of memory T cells with purified dendritic cells (DC) prevents their reversion to G0 and maintains their characteristic potent effector responses. Blockade of two TNFR molecules, CD70 and 4-1BB reverses the protection provided by DC. Furthermore, direct stimulation of the CD70 receptor on T cells, CD27, also maintains T cells in the memory state in the absence of any other cells. These results were verified with memory T cells generated against lymphocytic choriomeningitis virus (LCMV) and identified by MHC-peptide tetramers, which can identify individual antigen-specific cells. 3. CD27-deficient mice, memory T cells responsive to vesicular stomatitis virus (VSV) have difficulty maintaining their G1 state. 4. Ligation of CD27 activated phosphatidylinositol-3 kinase (PI3K) and Akt. This in turn resulted in phosphorylation of the quiescence transcription factor FOXO1 and its export from the nucleus (deactivation). Notably, whereas freshly isolated naive T cells expressed a substantial amount of FOXO1, levels were markedly reduced in memory T cells. These results account for the requirement for CD27 signaling in memory T cell maintenance. PI3K/Akt activate mammalian target of rapamycin (mTOR), which plays a vital role in protein translation and cell cycle progression. FOXO1 is important for maintaining cells in a quiescent state (i.e. in G0). Based upon this work, we have generated CD70-deficient mice. We have found that these animals have a blunted primary response to LCMV and fail to clear the virus normally. In contrast, the generation of memory T cells appears to be relatively normal. We have found that the loss of CD70 reduces the primary immune response to virus, but does not play a non-redundant role in generation of T cell memory. Furthermore, these animals allowed us to study CD70 expression on dendritic cells (DC). We found that the levels are virtually undetectable on resting DC, but that the protein is there and is recycling in and out of the cell. Furthermore, the level of CD70 is repressed at the transcriptional level by interaction with CD27. CD70 levels are repressed until T cells need to become activated. When they are, CD27 is reduced, resulting in consequent upregulation of CD70. Thus, there is dynamic regulation of CD70 due to interplay with its receptor. In parallel studies with wild type animals, we repeatedly observed a small population of cells in the lymph nodes and spleen that expressed an alpha/beta T cell receptor (TCR), CD11c, and MHC class II (the latter two being hallmarks of conventional DC). These cells, which we call TDC, have been characterized extensively at the cDNA and protein level. TDC, which can be either CD4+ or CD8+, but not both, require a thymus to develop like conventional T cells. However, like DC, they require FLT3L to develop. cDNA microarray analysis found that TDC express many genes heretofore found uniquely in T cells (TCR and CD3 genes) or DC (PU.1 and Zbtb46), but not both, as well as a distinctive signature of cytotoxicity-related genes. Unlike other T cell subsets characterized as innate, TDC had a polyclonal TCR repertoire and responded to cognate antigen. However, they differed from conventional T cells in that they did not require help from antigen-presenting cells. Strikingly, TDC responded to TLR-mediated stimulation by producing IL-12 and processed and presented antigen to MHCII-restricted T cells. TDC, as defined by surface phenotype and the ability to produce IL-12 when stimulated via TLRs, were identified in human peripheral blood as well. TDC, therefore, represent a heretofore unappreciated cell subset that combines key features of both innate and adaptive immune cells. We are currently working on strategies to specifically delete TDC so that we can study their function in vivo.